Non-protein stabilized clostridial toxin pharmaceutical compositions

ABSTRACT

A Clostridial toxin pharmaceutical composition comprising a Clostridial toxin, such as a botulinum toxin, wherein the Clostridial toxin present in the pharmaceutical composition is stabilized by a non-protein excipient such as a polyvinylpyrrolidone, a disaccharides, a trisaccharide, a polysaccharide, an alcohol, a metal, an amino acid, a surfactant and/or a polyethylene glycol.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.11/524,683, filed Sep. 21, 2006, which claims priority to relatedprovisional application No. 60/725,126, filed Oct. 6, 2005, and theentire content of each application is incorporated herein by reference.

BACKGROUND

The present invention relates to Clostridial toxin pharmaceuticalcompositions. In particular, the present invention relates toClostridial toxin pharmaceutical compositions with a non-proteinexcipient which functions to stabilize the Clostridial toxin (such as abotulinum toxin) present in the pharmaceutical composition.

A pharmaceutical composition is a formulation which contains at leastone active ingredient (such as a Clostridial toxin) as well as, forexample, one or more excipients, buffers, carriers, stabilizers,preservatives and/or bulking agents, and is suitable for administrationto a patient to achieve a desired diagnostic result or therapeuticeffect. The pharmaceutical compositions disclosed herein havediagnostic, therapeutic and/or research utility.

For storage stability and convenience of handling, a pharmaceuticalcomposition can be formulated as a lyophilized (i.e. freeze dried) orvacuum dried powder which can be reconstituted with a suitable fluid,such as saline or water, prior to administration to a patient.Alternately, the pharmaceutical composition can be formulated as anaqueous solution or suspension. A pharmaceutical composition can containa proteinaceous active ingredient. Unfortunately, a protein activeingredient can be very difficult to stabilize (i.e. maintained in astate where loss of biological activity is minimized), resultingtherefore in a loss of protein and/or loss of protein activity duringthe formulation, reconstitution (if required) and during the period ofstorage prior to use of a protein containing pharmaceutical composition.Stability problems can occur because of protein denaturation,degradation, dimerization, and/or polymerization. Various excipients,such as albumin and gelatin have been used with differing degrees ofsuccess to try and stabilize a protein active ingredient present in apharmaceutical composition. Additionally, cryoprotectants such asalcohols have been used to reduce protein denaturation under thefreezing conditions of lyophilization.

Protein Excipients

Various proteins such as albumin and gelatin have been used to stabilizea botulinum toxin present in a pharmaceutical composition. Albumins aresmall, abundant plasma proteins. Human serum albumin has a molecularweight of about 69 kiloDaltons (kD) and has been used as a non-activeingredient in a pharmaceutical composition where it can serve as a bulkcarrier and stabilizer of certain protein active ingredients present ina pharmaceutical composition.

The stabilization function of albumin in a pharmaceutical compositioncan be present both during the multi-step formulation of thepharmaceutical composition and upon the later reconstitution of theformulated pharmaceutical composition. Thus, stability can be impartedby albumin to a proteinaceous active ingredient in a pharmaceuticalcomposition by, for example, (1) reducing adhesion (commonly referred toas “stickiness”) of the protein active ingredient to surfaces, such asthe surfaces of laboratory glassware, vessels, to the vial in which thepharmaceutical composition is reconstituted and to the inside surface ofa syringe used to inject the pharmaceutical composition. Adhesion of aprotein active ingredient to surfaces can lead to loss of activeingredient and to denaturation of the remaining retained protein activeingredient, both of which reduce the total activity of the activeingredient present in the pharmaceutical composition, and; (2) reducingdenaturation of the active ingredient which can occur upon preparationof a low dilution solution of the active ingredient.

As well as being able to stabilize a protein active ingredient in apharmaceutical composition, human serum albumin also has the advantageof generally negligible immunogenicity when injected into a humanpatient. A compound with an appreciable immunogenicity can cause theproduction is of antibodies against it which can lead to an anaphylacticreaction and/or to the development of drug resistance, with the diseaseor disorder to be treated thereby becoming potentially refractory to thepharmaceutical composition which has an immunogenic component.

Recombinant albumin has been proposed as a stabilizer in a botulinumtoxin pharmaceutical composition. Thus, published U.S. patentapplication number 2003 0118598 (Hunt) discloses uses of variousexcipients such as a recombinant albumin, collagen or a starch tostabilize a botulinum toxin present in a pharmaceutical composition.

Collagen is the most abundant protein in mammals comprising about onequarter of all protein in the body and it is the major constituent ofconnective tissues, such as skin, ligaments and tendons. Native collagenis a triple helix of three high molecular weight proteins. Each of thethree protein chains comprising the collagen helix has more than 1400amino acids. At least twenty five distinct types of collagens have beenidentified in humans.

Collagen has been used cosmetically as a filler material to treat ofskin contour problems, such as to smooth smile line grooves and frownlines, folds between the eyebrows, wrinkles in the corners of the eyesand fine vertical creases above and below the lips. Collagen is alsouseful in smoothing certain post-surgical traumatic or acne scarring andviral pock marks, such as chicken pox marks. For such purposes thecollagen is injected into the dermis to raise the skin.

Gelatin can be obtained by the hydrolysis of collagen. Gelatin has beenused in some protein active ingredient pharmaceutical compositions as analbumin substitute. Notably, gelatin is a animal derived protein andtherefore carries the same risk of potential infectivity which may bepossessed by human serum albumin. Chinese patent CN 1215084 is discussesan albumin free botulinum toxin type A formulated with native gelatin (acollagen hydrolysate), an animal derived protein, dextran and sucrose.U.S. Pat. No. 6,087,327 also discloses a composition of botulinum toxintypes A and B formulated with native gelatin.

Unfortunately, despite their known stabilizing effects, significantdrawbacks exist to the use of protein excipients, such as albumin orgelatin, in a pharmaceutical composition. For example albumin andgelatin are expensive and increasingly difficult to obtain. Furthermore,blood products or animal derived products such as albumin and gelatin,when administered to a patient can subject the patient to a potentialrisk of receiving blood borne pathogens or infectious agents. Thus, itis known that the possibility exists that the presence of an animalderived protein excipient in a pharmaceutical composition can result ininadvertent incorporation of infectious elements into the pharmaceuticalcomposition. For example, it has been reported that use of human serumalbumin may transmit prions into a pharmaceutical composition. A prionis a proteinaceous infectious particle which is hypothesized to arise asan abnormal conformational isoform from the same nucleic acid sequencewhich makes the normal protein. It has been further hypothesized thatinfectivity resides in a “recruitment reaction” of the normal isoformprotein to the prion protein isoform at a post translational level.Apparently the normal endogenous cellular protein is induced to misfoldinto a pathogenic prion conformation.

Thus, it is desirable to find a suitable excipient which can be used tostabilize the botulinum toxin present in a botulinum toxinpharmaceutical composition. Preferably, the botulinum toxin stabilizeris not a protein derived from an animal (i.e. mammalian) source.

Botulinum Toxin

The genus Clostridium has more than one hundred and twenty sevenspecies, grouped by morphology and function. The anaerobic, gram ispositive bacterium Clostridium botulinum produces a potent polypeptideneurotoxin, botulinum toxin, which causes a neuroparalytic illness inhumans and animals referred to as botulism. Clostridium botulinum andits spores are commonly found in soil and the bacterium can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A is the most lethal natural biological agent knownto man. About 50 picograms of botulinum toxin (purified neurotoxincomplex) type A is a LD₅₀ in mice. Interestingly, on a molar basis,botulinum toxin type A is 1.8 billion times more lethal than diphtheria,600 million times more lethal than sodium cyanide, 30 million times morelethal than cobrotoxin and 12 million times more lethal than cholera.Singh, Critical Aspects of Bacterial Protein Toxins, pages 63-84(chapter 4) of Natural Toxins II, edited by B. R. Singh et al., PlenumPress, New York (1976) (where the stated LD₅₀ of botulinum toxin type Aof 0.3 ng equals 1 U is corrected for the fact that about 0.05 ng ofBOTOX® equals 1 unit). One unit (U) of botulinum toxin is defined as theLD₅₀ upon intraperitoneal injection into female Swiss Webster miceweighing 18-20 grams each. In other words, one unit of botulinum toxinis the amount of botulinum toxin that kills 50% of a group of femaleSwiss Webster mice. Seven generally immunologically distinct botulinumneurotoxins have been characterized, these being respectively botulinumneurotoxin serotypes A, B, C₁, D, E, F, and G, each of which isdistinguished by neutralization with type-specific antibodies. Thedifferent serotypes of botulinum toxin vary in the animal species thatthey affect and in the severity and duration of the paralysis theyevoke. For example, it has been determined that botulinum toxin type Ais 500 times more potent, as measured by the rate of paralysis producedin the rat, than is botulinum toxin type B. Additionally, botulinumtoxin type B has been determined to be non-toxic in primates at a doseof 480 U/kg which is about 12 times the primate LD₅₀ for botulinum toxintype A. The botulinum toxins apparently bind with high affinity tocholinergic motor neurons, are translocated into the neuron and blockthe presynaptic release of acetylcholine.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletalmuscles. Botulinum toxin type A was approved by the U.S. Food and DrugAdministration in 1989 for the treatment of essential blepharospasm,strabismus and hemifacial spasm in patients over the age of twelve.Clinical effects of peripheral injection (i.e. intramuscular orsubcutaneous) botulinum toxin type A are usually seen within one week ofinjection, and often within a few hours after injection. The typicalduration of symptomatic relief (i.e. flaccid muscle paralysis) from asingle intramuscular injection of botulinum toxin type A can be aboutthree months to about six months.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. Botulinum toxin A is a zincendopeptidase which can specifically hydrolyze a peptide linkage of theintracellular, vesicle associated protein SNAP-25. Botulinum type E alsocleaves the 25 kiloDalton (kD) synaptosomal associated protein(SNAP-25), but targets different amino acid sequences within thisprotein, as compared to botulinum toxin type A. Botulinum toxin types B,D, F and G act on vesicle-associated protein (VAMP, also calledsynaptobrevin), with each serotype cleaving the protein at a differentsite. Finally, botulinum toxin type C₁ has been shown to cleave bothsyntaxin and SNAP-25. These differences in mechanism of action mayaffect the relative potency and/or duration of action of the variousbotulinum toxin serotypes.

Regardless of serotype, the molecular mechanism of toxin intoxicationappears to be similar and to involve at least three steps or stages. Inthe first step of the process, the toxin binds to the presynapticmembrane of the target neuron through a specific interaction between theheavy chain (H chain) and a cell surface receptor; the receptor isthought to be different for each serotype of botulinum toxin and fortetanus toxin. The carboxyl end segment of the H chain, H_(C), appearsto be important for targeting of the toxin to the cell surface.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This last step is thought to be mediated by the amino end segmentof the H chain, H_(N), which triggers a conformational change of thetoxin in response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxinthen translocates through the endosomal membrane into the cytosol.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the H and L chain. Theentire toxic activity of botulinum and tetanus toxins is contained inthe L chain of the holotoxin; the L chain is a zinc (Zn++) endopeptidasewhich selectively cleaves proteins essential for recognition and dockingof neurotransmitter-containing vesicles with the cytoplasmic surface ofthe plasma membrane, and fusion of the vesicles with the plasmamembrane. Tetanus neurotoxin, botulinum toxin B, D, F, and G causedegradation of synaptobrevin (also called vesicle-associated membraneprotein (VAMP)), a synaptosomal membrane protein. Most of the VAMPpresent at the cytosolic surface of the synaptic vesicle is removed as aresult of any one of these cleavage events. Each toxin specificallycleaves a different bond.

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD. Botulinumtoxins are released by Clostridial bacterium as complexes comprising the150 kD botulinum toxin protein molecule along with associated non-toxinproteins. Thus, the botulinum toxin type A complex can be produced byClostridial bacterium as 900 kD, 500 kD and 300 kD forms. botulinumtoxin types B and C₁ are apparently produced as only a 500 kD complex.botulinum toxin type D is produced as both 300 kD and 500 kD complexes.Finally, botulinum toxin types E and F are produced as onlyapproximately 300 kD complexes. The complexes (i.e. molecular weightgreater than about 150 kD) are believed to contain a non-toxinhemagglutinin protein and a non-toxin and non-toxic nonhemagglutininprotein. These two non-toxin proteins (which along with the botulinumtoxin molecule can comprise the relevant neurotoxin complex) may act toprovide stability against denaturation to the botulinum toxin moleculeand protection against digestive acids when toxin is ingested.Additionally, it is possible that the larger (greater than about 150 kDmolecular weight) botulinum toxin complexes may result in a slower rateof diffusion of the botulinum toxin away from a site of intramuscularinjection of a botulinum toxin complex. The toxin complexes can bedissociated into toxin protein and hemagglutinin proteins by treatingthe complex with red blood cells at pH 7.3. The toxin protein has amarked instability upon removal of the hemagglutinin protein.

All the botulinum toxin serotypes are made by Clostridium botulinumbacteria as inactive single chain proteins which must be cleaved ornicked by proteases to become neuroactive. The bacterial strains thatmake botulinum toxin serotypes A and G possess endogenous proteases andserotypes A and G can therefore be recovered from bacterial cultures inpredominantly their active form. In contrast, botulinum toxin serotypesC₁, D, and E are synthesized by nonproteolytic strains and are thereforetypically unactivated when recovered from culture. Serotypes B and F areproduced by both proteolytic and nonproteolytic strains and thereforecan be recovered in either the active or inactive form. However, eventhe proteolytic strains that produce, for example, the botulinum toxintype B serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP and glutamate.

High quality crystalline botulinum toxin type A can be produced from theHall A strain of Clostridium botulinum with characteristics of ≧3×10⁷U/mg, an A₂₆₀/A₂₇₈ of less than 0.60 and a distinct pattern of bandingon gel electrophoresis. The known Schantz process can be used to obtaincrystalline botulinum toxin type A, as set forth in Schantz, E. J., etal, Properties and use of Botulinum toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56: 80-99 (1992). Generally, thebotulinum toxin type A complex can be isolated and purified from ananaerobic fermentation by cultivating Clostridium botulinum type A in asuitable medium. Raw toxin can be harvested by precipitation withsulfuric acid and concentrated by ultramicrofiltration. Purification canbe carried out by dissolving the acid precipitate in calcium chloride.The toxin can then be precipitated with cold ethanol. The precipitatecan be dissolved in sodium phosphate buffer and centrifuged. Upon dryingthere can then be obtained approximately 900 kD crystalline botulinumtoxin type A complex with a specific potency of 3×10⁷ LD₅₀ U/mg orgreater. This known process can also be used, upon separation out of thenon-toxin proteins, to obtain pure botulinum toxins, such as forexample: purified botulinum toxin type A with an approximately 150 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; purified botulinum toxin type B with an approximately 156 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater, and; purified botulinum toxin type F with an approximately 155kD molecular weight with a specific potency of 1-2×10⁷ LD₅₀ U/mg orgreater.

Botulinum toxins and toxin complexes can be obtained from, for example,List Biological Laboratories, Inc., Campbell, Calif.; the Centre forApplied Microbiology and Research, Porton Down, U.K.; Wako (Osaka,Japan), as well as from Sigma Chemicals of St Louis, Mo. Commerciallyavailable botulinum toxin containing pharmaceutical compositions includeBOTOX® (Botulinum toxin type A neurotoxin complex with human serumalbumin and sodium chloride) available from Allergan, Inc., of Irvine,Calif. in 100 unit vials as a lyophilized powder to be reconstitutedwith 0.9% sodium chloride before use), Dysport® (Clostridium botulinumtype A toxin haemagglutinin complex with human serum albumin and lactosein the formulation), available from Ipsen Limited, Berkshire, U.K. as apowder to be reconstituted with 0.9% sodium chloride before use), andMyoBloc™ (an injectable solution comprising botulinum toxin type B,human serum albumin, sodium succinate, and sodium chloride at about pH5.6, available from Solstice Neurosciences, Inc., South San Francisco,Calif.).

The success of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes.Additionally, pure botulinum toxin has been used to treat humans. Seee.g. Kohl A., et al., Comparison of the effect of botulinum toxin A(Botox (R)) with the highly-purified neurotoxin (NT 201) in the extensordigitorum brevis muscle test, Mov Disord 2000; 15(Suppl 3):165. Hence, apharmaceutical composition can be prepared using a pure botulinum toxin.

The type A botulinum toxin is known to be soluble in dilute aqueoussolutions at pH 4-6.8. At pH above about 7 the stabilizing nontoxicproteins dissociate from the neurotoxin, resulting in a gradual loss oftoxicity, particularly as the pH and temperature rise. Schantz E. J., etal Preparation and characterization of botulinum toxin type A for humantreatment (in particular pages 44-45), being chapter 3 of Jankovic, J.,et al, Therapy with Botulinum Toxin, Marcel Dekker, Inc (1994).

European patent EP1112082 (“Stable liquid formulations of botulinumtoxin”), issued Jul. 31, 2002 claims a stable liquid pharmaceuticalbotulinum toxin formulation comprising a buffer (pH 5-6) and a botulinumtoxin, wherein the toxin formulation is stable as a liquid for at leastone year at temperatures between 0-10 C or at least 6 months attemperatures between 10 and 30 C. Such a botulinum toxin pharmaceuticalformulation (an embodiment of which is sold commercially under thetradename MyoBloc® or NeuroBloc® by Solstice Neurosciences, Inc., of SanDiego, Calif.) is prepared as a liquid solution (no lyophilization orvacuum drying is carried out) which does not require reconstitutionbefore use.

U.S. Pat. No. 5,512,547 (Johnson et al) entitled “PharmaceuticalComposition of Botulinum Neurotoxin and Method of Preparation” issuedApr. 30, 1996 and claims a pure botulinum type A formulation comprisingalbumin and trehalose, storage stable at 37 degrees C.

U.S. Pat. NO. 5,756,468 (Johnson et al) issued May 26, 1998(“Pharmaceutical Compositions of Botulinum Toxin or Botulinum Neurotoxinand Method of Preparation”), and claims a lyophilized botulinum toxinformulation comprising a thioalkyl, albumin and trehalose which can bestored between 25 degrees C. and 42 degrees C.

U.S. Pat. No. 5,696,077 (Johnson et al) entitled “PharmaceuticalComposition Containing Botulinum B Complex” issued Dec. 9, 1997 andclaims a freeze dried, sodium chloride-free botulinum type B complexformation comprising a type B complex and a protein excipient.

Goodnough M. C., et al., Stabilization of botulinum toxin type A duringlyophilization, Appl Environ Microbiol 1992; 58(10):3426-3428, and;Goodnough M. C., et al., Recovery of type-A botulinal toxin followinglyophilization, Acs Symposium Series 1994; 567(−):193-203, discloses.

Chinese patent application CN 1215084A discusses an albumin freebotulinum toxin type A formulated with gelatin, an animal derivedprotein. U.S. Pat. No. 6,087,327 also discloses a composition ofbotulinum toxin types A and B formulated with gelatin. Theseformulations therefore do not eliminate the risk of transmitting ananimal protein derived or accompanying infectious element.

It has been reported that BoNt/A has been used in various clinicalsettings, including as follows:

(1) about 75-125 units of BOTOX®¹ per intramuscular injection (multiplemuscles) to treat cervical dystonia; ¹ Available from Allergan, Inc., ofIrvine, Calif. under the tradename BOTOX®.

(2) 5-10 units of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) (5 units injected intramuscularly into the procerusmuscle and 10 units injected intramuscularly into each corrugatorsupercilii muscle);

(3) about 30-80 units of BOTOX® to treat constipation by intrasphincterinjection of the puborectalis muscle;

(4) about 1-5 units per muscle of intramuscularly injected BOTOX® totreat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

(5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

(6) to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows:

(a) flexor digitorum profundus: 7.5 U to 30 U

(b) flexor digitorum sublimus: 7.5 U to 30 U

(c) flexor carpi ulnaris: 10 U to 40 U

(d) flexor carpi radialis: 15 U to 60 U

(e) biceps brachii: 50 U to 200 U. Each of the five indicated muscleshas been injected at the same treatment session, so that the patientreceives from 90 U to 360 U of upper limb flexor muscle BOTOX® byintramuscular injection at each treatment session.

(7) to treat migraine, pericranial injected (injected symmetrically intoglabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX®has showed significant benefit as a prophylactic treatment of migrainecompared to vehicle as measured by decreased measures of migrainefrequency, maximal severity, associated vomiting and acute medicationuse over the three month period following the 25 U injection.

It is known that botulinum toxin type A can have an efficacy for up to12 months (European J. Neurology 6 (Supp 4): S111-S1150:1999), and insome circumstances for as long as 27 months. The Laryngoscope109:1344-1346:1999. However, the usual duration of an intramuscularinjection of Botox® is typically about 3 to 4 months.

The success of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes.Additionally, pure botulinum toxin has been used in humans. See e.g.Kohl A., et al., Comparison of the effect of botulinum toxin A (Botox(R)) with the highly-purified neurotoxin (NT 201) in the extensordigitorum brevis muscle test, Mov Disord 2000; 15(Suppl 3):165. Hence, apharmaceutical composition can be prepared using a pure botulinum toxin.

The botulinum toxin molecule (about 150 kDa), as well as the botulinumtoxin complexes (about 300-900 kDa), such as the toxin type A complexare also extremely susceptible to denaturation due to surfacedenaturation, heat, and alkaline conditions. Inactivated toxin formstoxoid proteins which may be immunogenic. The resulting antibodies canrender a patient refractory to toxin injection.

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) are dependant, at least inpart, upon their three dimensional conformation. Thus, botulinum toxintype A is detoxified by heat, various chemicals surface stretching andsurface drying. Additionally, it is known that dilution of the toxincomplex obtained by the known culturing, fermentation and purificationto the much, much lower toxin concentrations used for pharmaceuticalcomposition formulation results in rapid detoxification of the toxinunless a suitable stabilizing agent is present. Dilution of the toxinfrom milligram quantities to a solution containing nanograms permilliliter presents significant difficulties because of the rapid lossof specific toxicity upon such great dilution. Since the toxin may beused months or years after the toxin containing pharmaceuticalcomposition is formulated, the toxin must be stabilized with astabilizing agent. To date, the only successful stabilizing agent forthis purpose has been the animal derived proteins human serum albuminand gelatin.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxintype A complex, human serum albumin, and sodium chloride packaged insterile, vacuum-dried form. The botulinum toxin type A is made from aculture of the Hall strain of Clostridium botulinum grown in a mediumcontaining N-Z amine and yeast extract. The botulinum toxin type Acomplex is purified from the culture solution by a series of acidprecipitations to a crystalline complex consisting of the active highmolecular weight toxin protein and an associated hemagglutinin protein.The crystalline complex is re-dissolved in a solution containing salineand albumin and sterile filtered (0.2 microns) prior to vacuum-drying.BOTOX® can be reconstituted with sterile, non-preserved saline prior tointramuscular injection. Each vial of BOTOX® contains about 100 units(U) of Clostridium botulinum toxin type A complex, 0.5 milligrams ofhuman serum albumin and 0.9 milligrams of sodium chloride in a sterile,vacuum-dried form without a preservative.

is To reconstitute vacuum-dried BOTOX® sterile normal saline without apreservative (0.9% Sodium Chloride injection) is used by drawing up theproper amount of diluent in the appropriate size syringe. Since BOTOX®is denatured by bubbling or similar violent agitation, the diluent isgently injected into the vial. For sterility reasons, BOTOX® should beadministered within four hours after reconstitution. During this timeperiod, reconstituted BOTOX® is stored in a refrigerator (2° to 8° C.).Reconstituted BOTOX® is clear, colorless and free of particulate matter.The vacuum-dried product is stored in a freezer at or below −5° C.

It has been reported that a suitable alternative to human serum albuminas a botulinum toxin stabilizer may be another protein or alternativelya low molecular weight (non-protein) compound. Carpender et al.,Interactions of Stabilizing Additives with Proteins DuringFreeze-Thawing and Freeze-Drying, International Symposium on BiologicalProduct Freeze-Drying and Formulation, 24-26 Oct. 1990; Karger (1992),225-239.

Many substances commonly used as carriers and bulking agents inpharmaceutical compositions have proven to be unsuitable as non-proteinexcipients to stabilize the botulinum toxin present in a pharmaceuticalcomposition. For example, the disaccharide cellobiose has been found tobe unsuitable as a botulinum toxin stabilizer. Thus, it is known thatthe use of cellobiose as an excipient in conjunction with albumin andsodium chloride results in a much lower level of toxicity (10% recovery)after lyophilization of crystalline botulinum toxin type A with theseexcipients, as compared to the toxicity after lyophilization with onlyhuman serum albumin (>75% to >90% recovery). Goodnough et al.,Stabilization of Botulinum Toxin Type A During Lyophilization, App &Envir. Micro. 58 (10) 3426-3428 (1992).

Furthermore, saccharides, including polysaccharides, are in general poorcandidates to serve as protein stabilizers. Thus, it is known that apharmaceutical composition containing a protein active ingredient isinherently unstable if the protein formulation comprises a saccharide(such as glucose or a polymer of glucose) or carbohydrates becauseproteins and glucose are known to interact together and to undergo thewell-described Maillard reaction, due to the reducing nature of glucoseand glucose polymers. Much work has been dedicated to mostlyunsuccessful attempts at preventing this protein-saccharide reaction by,for example, reduction of moisture or use of non-reducing sugars.Significantly, the degradative pathway of the Maillard reaction canresult in a therapeutic insufficiency of the protein active ingredient.A pharmaceutical formulation comprising protein and a reducingsaccharide, carbohydrate or sugar, such as a glucose polymer, istherefore inherently unstable and cannot be stored for a long period oftime without significant loss of the active ingredient protein's desiredbiological activity.

Particular high molecular weight polysaccharides (starches), such ashetastarch, have been proposed as stabilizers of the botulinum toxinpresent in a botulinum toxin pharmaceutical composition. See e.g.European patent EP 1 253 932, issued Apr. 27, 2005.

Notably, one of the reasons albumin or gelatin can function effectivelyas a stabilizer of a protein active ingredient in a pharmaceuticalcomposition is because being proteins these stabilizers do not undergothe Maillard reaction with the protein active ingredient in apharmaceutical composition. Hence, one would expect to find and to lookfor a substitute for these protein excipients used to stabilize thebotulinum toxin present in a botulinum toxin pharmaceutical compositionamongst other proteins.

Unique characteristics of botulinum toxin and its formulation into a issuitable pharmaceutical composition constrain and hinder and render thesearch for a replacement for a protein stabilizer in a botulinum toxincontaining pharmaceutical formulations problematic. Examples of four ofthese unique characteristics follow.

First, botulinum toxin is a relatively large protein for incorporationinto a pharmaceutical formulation (the molecular weight of the botulinumtoxin type A complex is 900 kD) and is therefore is inherently fragileand labile. The size of the toxin complex makes it much more friable andlabile than smaller, less complex proteins, thereby compounding theformulation and handling difficulties if toxin stability is to bemaintained. Hence, a botulinum toxin stabilizer must be able to interactwith the toxin in a manner which does not denature, fragment orotherwise detoxify the toxin molecule or cause disassociation of thenon-toxin proteins present in the toxin complex.

Second, as the most lethal known biological product, exceptional safety,precision, and accuracy is called for at all steps of the formulation ofa botulinum toxin containing pharmaceutical composition. Thus, abotulinum toxin stabilizer should not itself be toxic or difficult tohandle so as to not exacerbate the already extremely stringent botulinumtoxin containing pharmaceutical composition formulation requirements.

Third, since botulinum toxin was the first microbial toxin to beapproved (by the FDA in 1989) for injection for the treatment of humandisease, specific protocols had to be developed and approved for theculturing, bulk production, formulation into a pharmaceutical and use ofbotulinum toxin. Important considerations are toxin purity and dose forinjection. The production by culturing and the purification must becarried out so that the toxin is not exposed to any substance that mightcontaminate the final product in even trace amounts and cause unduereactions in the patient. These restrictions require culturing insimplified medium without the use of animal meat products andpurification by procedures not involving synthetic solvents or resins.Preparation of toxin using enzymes, various exchangers, such as thosepresent in chromatography columns and synthetic solvents can introducecontaminants and are therefore excluded from preferred formulationsteps. Furthermore, botulinum toxin type A is readily denatured attemperatures above 40 degrees C., loses toxicity when bubbles form atthe air/liquid interface, and denatures in the presence of nitrogen orcarbon dioxide.

Fourth, particular difficulties exist to stabilize botulinum toxin typeA, because type A consists of a toxin molecule of about 150 kD innoncovalent association with nontoxin proteins weighing about 750 kD.The nontoxin proteins are believed to preserve or help stabilize thesecondary and tertiary structures upon which toxicity is dependant.Procedures or protocols applicable to the stabilization of nonproteinsor to relatively smaller proteins are not applicable to the problemsinherent with stabilization of the botulinum toxin complexes, such asthe 900 kD botulinum toxin type A complex. Thus while from pH 3.5 to 6.8the type A toxin and non toxin proteins are bound togethernoncovalently, under slightly alkaline conditions (pH>7.1) the verylabile toxin is released from the toxin complex. As set forthpreviously, pure botulinum toxin (i.e. the 150 kD molecule) has beenproposed as the active ingredient in a pharmaceutical composition.

In light of the unique nature of botulinum toxin and the requirementsset forth above, the probability of finding a suitable non-proteinstabilizer for the protein stabilizers used in botulinum toxincontaining pharmaceutical compositions must realistically be seen toapproach zero. Prior to the present invention, only the animal derivedproteins, human serum albumin and gelatin, had been known to haveutility as suitable stabilizers of the botulinum toxin present in apharmaceutical formulation. Thus, albumin, by itself or with one or moreadditional substances such as sodium phosphate or sodium citrate, isknown to permit high recovery of toxicity of botulinum toxin type Aafter lyophilization. Unfortunately, as already set forth, human serumalbumin, as a pooled blood product, can, at least potentially, carryinfectious or disease causing elements when present in a pharmaceuticalcomposition. Indeed, any animal product or protein such as human serumalbumin or gelatin can also potentially contain pyrogens or othersubstances that can cause adverse reactions upon injection into apatient.

What is needed therefore is a Clostridial toxin pharmaceuticalcompositions wherein the Clostridial toxin (such as a botulinum toxin)is stabilized by a non-protein excipient.

SUMMARY

The present invention meets this need and provides a botulinum toxinpharmaceutical composition which is stabilized by a non-proteinexcipient.

Definitions

As used herein, the words or terms set forth below have the followingdefinitions.

“About” means that the item, parameter or term so qualified encompassesa range of plus or minus ten percent above and below the value of thestated item, parameter or term.

“Administration”, or “to administer” means the step of giving (i.e.administering) a pharmaceutical composition to a subject. Thepharmaceutical compositions disclosed herein are “locally administered”by e.g. intramuscular (i.m.), intradermal, subcutaneous administration,intrathecal administration, intraperitoneal (i.p.) administration,topical (transdermal) and implantation (i.e. of a slow-release devicesuch as polymeric implant or miniosmotic pump) routes of administration.

“Animal protein free” means the absence of blood derived, blood pooledand other animal derived products or compounds. “Animal” means a mammal(such as a human), bird, reptile, fish, insect, spider or other animalspecies. “Animal” excludes microorganisms, such as bacteria. Thus, ananimal protein free pharmaceutical composition within the scope of myinvention can include a Clostridial neurotoxin. For example, an animalprotein free pharmaceutical composition means a pharmaceuticalcomposition which is either substantially free or essentially free orentirely free of a serum derived albumin, gelatin and other animalderived proteins, such as immunoglobulins. An example of an animalprotein free pharmaceutical composition is a pharmaceutical compositionwhich comprises or which consists of a botulinum toxin (as the activeingredient) and a suitable polysaccharide as a stabilizer or excipient.

“Botulinum toxin” means a neurotoxin produced by Clostridium botulinum,as well as a botulinum toxin (or the light chain or the heavy chainthereof) made recombinantly by a non-Clostridial species. The phrase“botulinum toxin”, as used herein, encompasses the botulinum toxinserotypes A, B, C, D, E, F and G. Botulinum toxin, as used herein, alsoencompasses both a botulinum toxin complex (i.e. the 300, 600 and 900kDa complexes) as well as the purified botulinum toxin (i.e. about 150kDa). “Purified botulinum toxin” is defined as a botulinum toxin that isisolated, or substantially isolated, from other proteins, includingproteins that form a botulinum toxin complex. A purified botulinum toxinmay be greater than 95% pure, and preferably is greater than 99% pure.The botulinum C₂ and C₃ cytotoxins, not being neurotoxins, are excludedfrom the scope of the present invention.

“Clostridial neurotoxin” means a neurotoxin produced from, or native to,a Clostridial bacterium, such as Clostridium botulinum, Clostridiumbutyricum or Clostridium beratti, as well as a Clostridial neurotoxinmade recombinantly by a non-Clostridial species.

“Entirely free (i.e. “consisting of” terminology) means that within thedetection range of the instrument or process being used, the substancecannot be detected or its presence cannot be confirmed.

“Essentially free” (or “consisting essentially of”) means that onlytrace amounts of the substance can be detected.

“Modified botulinum toxin” means a botulinum toxin that has had at leastone of its amino acids deleted, modified, or replaced, as compared to anative botulinum toxin. Additionally, the modified botulinum toxin canbe a recombinantly produced neurotoxin, or a derivative or fragment of arecombinantly made neurotoxin. A modified botulinum toxin retains atleast one biological activity of the native botulinum toxin, such as,the ability to bind to a botulinum toxin receptor, or the ability toinhibit neurotransmitter release from a neuron. One example of amodified botulinum toxin is a botulinum toxin that has a light chainfrom one botulinum toxin serotype (such as serotype A), and a heavychain from a different botulinum toxin serotype (such as serotype B).Another example of a modified botulinum toxin is a botulinum toxincoupled to a neurotransmitter, such as substance P.

“Pharmaceutical composition” means a formulation in which an activeingredient can be a Clostridial neurotoxin, such as a botulinum toxin.is The word “formulation” means that there is at least one additionalingredient in the pharmaceutical composition besides a Clostridialneurotoxin active ingredient. A pharmaceutical composition is thereforea formulation which is suitable for diagnostic or therapeuticadministration (i.e. by intramuscular or subcutaneous injection or byinsertion of a depot or implant) to a subject, such as a human patient.The pharmaceutical composition can be: in a lyophilized or vacuum driedcondition; a solution formed after reconstitution of the lyophilized orvacuum dried pharmaceutical composition with saline or water, or; as asolution which does not require reconstitution. The neurotoxin activeingredient can be one of the botulinum toxin serotypes A, B, C₁, D, E, For G or a tetanus toxin, all of which can be made natively byClostridial bacteria. As stated, a pharmaceutical composition can beliquid or solid, for example vacuum-dried. The constituent ingredientsof a pharmaceutical composition can be included in a single composition(that is all the constituent ingredients, except for any requiredreconstitution fluid, are present at the time of initial compounding ofthe pharmaceutical composition) or as a two-component system, forexample a vacuum-dried composition reconstituted with a diluent such assaline which diluent contains an ingredient not present in the initialcompounding of the pharmaceutical composition. A two-component systemprovides the benefit of allowing incorporation of ingredients which arenot sufficiently compatible for long-term shelf storage with the firstcomponent of the two component system. For example, the reconstitutionvehicle or diluent may include a preservative which provides sufficientprotection against microbial growth for the use period, for exampleone-week of refrigerated storage, but is not present during the two-yearfreezer storage period during which time it might degrade the toxin.Other ingredients, which may not be compatible with a Clostridial toxinor other ingredients for long periods of time, may be incorporated inthis manner; that is, added in a second vehicle (i.e. in thereconstitution fluid) at the approximate time of use.

“Stabilizer” (or “primary stabilizer”) is a chemical agent that assiststo preserve or maintain the biological structure (i.e. the threedimensional conformation) and/or biological activity of a protein (suchas a Clostridial neurotoxin, such as a botulinum toxin). The stabilizersused herein are non-proteins. The primary stabilizer can be a syntheticagent that would not produce an immunogenic response (or produces anattenuated immune response) in a subject receiving a compositioncontaining the primary stabilizer. Additional stabilizers may also beincluded in a pharmaceutical composition. These additional or secondarystabilizers may be used alone or in combination with the primarystabilizers. Exemplary secondary stabilizers include, but are notlimited to non-oxidizing amino acid derivatives (such as a tryptophanderivate, such as N-acetyl-tryptophan (“NAT”)), caprylate (i.e. sodiumcaprylate), a polysorbate (i.e. P80), amino acids, and divalent metalcations such as zinc. A pharmaceutical composition can also includepreservative agents such as benzyl alcohol, benzoic acid, phenol,parabens and sorbic acid.

“Stabilizing”, “stabilizes”, or “stabilization” mean that apharmaceutical active ingredient (“PAI”) retains at least 20% and up to100% of its biological activity (which can be assessed as potency or astoxicity by an in vivo LD₅₀ or ED₅₀ measure) in the presence of acompound which is stabilizing, stabilizes or which providesstabilization to the PAI. For example, upon (1) preparation of serialdilutions from a bulk or stock solution, or (2) upon reconstitution withsaline or water of a lyophilized, or vacuum dried botulinum toxincontaining pharmaceutical composition which has been stored at or belowabout −2 degrees C. for between six months and four years, or (3) for anaqueous solution botulinum toxin containing pharmaceutical compositionwhich has been stored at between about 2 degrees and about 8 degrees C.for from six months to four years, the botulinum toxin present in thereconstituted or aqueous solution pharmaceutical composition has (in thepresence of a compound which is stabilizing, stabilizes or whichprovides stabilization to the PAI) greater than about 20% and up toabout 100% of the potency or toxicity that the biologically activebotulinum toxin had prior to being incorporated into the pharmaceuticalcomposition.

“Substantially free” means present at a level of less than one percentby weight of the pharmaceutical composition.

“Therapeutic formulation” means a formulation can be used to treat andthereby alleviate a disorder or a disease, such as a disorder or adisease characterized by hyperactivity (i.e. spasticity) of a peripheralmuscle.

The botulinum toxin can be present as a botulinum toxin complex (i.e. asan approximately 300 to about 900 kiloDalton complex depending upon theparticular botulinum toxin serotype) or the botulinum toxin can be ispresent as a pure or purified botulinum toxin (i.e. as the botulinumtoxin molecule of about 150 kiloDaltons).

The pharmaceutical compositions disclosed herein can have a pH ofbetween about 5 and 7.3 when reconstituted or upon injection.

My invention can be practiced utilizing a composition that comprises abotulinum toxin type A. In other embodiments of the invention, theforegoing methods may be practiced with a composition that comprisesbotulinum toxin type B. In further embodiments of the invention, themethods may be practiced with a composition that comprises a pluralityof botulinum toxin serotypes, such as botulinum toxin serotypes selectedfrom the group consisting of botulinum toxin serotypes A, B, C₁, D, E, Fand G. In certain embodiments of the invention, purified botulinumtoxins may be used. In other embodiments, modified botulinum toxins maybe used.

In yet additional embodiments of the invention, the compositions used inthe foregoing methods can be administered intramuscularly to thepatient. In other embodiments, the compositions can be administeredsubcutaneously and/or intrathecally.

My invention encompasses a pharmaceutical composition comprising (orconsisting of or consisting essentially of) a botulinum toxin and apolyvinylpyrrolidone. The botulinum toxin is a biologically activebotulinum toxin and the botulinum toxin is selected from the groupconsisting of the botulinum toxins types A, B, C, D, E, F and G.Preferably, the botulinum toxin is a botulinum toxin type A. A functionof the polyvinylpyrrolidone present in the pharmaceutical composition isto stabilize the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin, andbetween about 5 grams and about 20 grams of a polyvinylpyrrolidone foreach about 100 units of the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient,and a polyvinylpyrrolidone, wherein the potency of the botulinum toxinis at least about 40% of the theoretical maximum potency of thebotulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient,and a polyvinylpyrrolidone, wherein the potency of the botulinum toxinis at least about 50% of the theoretical maximum potency of thebotulinum toxin.

My invention also encompasses a pharmaceutical composition is comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, apolyvinylpyrrolidone, and a disaccharide, wherein the potency of thebotulinum toxin is at least about 40% of the theoretical maximum potencyof the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, apolyvinylpyrrolidone, and a disaccharide, wherein the potency of thebotulinum toxin is at least about 50% of the theoretical maximum potencyof the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, apolyvinylpyrrolidone, and a disaccharide, wherein the potency of thebotulinum toxin is at least about 60% of the theoretical maximum potencyof the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, apolyvinylpyrrolidone, and a disaccharide, wherein the potency of thebotulinum toxin is at least about 70% of the theoretical maximum potencyof the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, apolyvinylpyrrolidone, and a polyethylene glycol, wherein the potency ofthe botulinum toxin is at least about 40% of the theoretical maximumpotency of the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, acompound selected from the group consisting of a first monosaccharide, afirst disaccharide, a first trisaccharide, and a first alcohol made byreducing the first monosaccharide, and a compound selected from thegroup of compounds consisting of a polyethylene glycol, a secondmonosaccharide, a second disaccharide, a second trisaccharide, a metal,a second alcohol, and an amino acid, wherein the second monosaccharide,the second disaccharide and the second trisaccharide are different fromrespectively the first monosaccharide, the first disaccharide, and thefirst trisaccharide, wherein the potency of the botulinum toxin at leastabout 40% of the theoretical maximum potency of the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, apolyethylene glycol, and a compound selected from the group of compoundsconsisting of a monosaccharide, a disaccharide, a trisaccharide, ametal, an alcohol, and an amino acid, wherein the potency of thebotulinum toxin is at least about 20% of the theoretical maximum potencyof the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, apolyethylene glycol, and a compound selected from the group of compoundsconsisting of a monosaccharide, a disaccharide, a trisaccharide, ametal, an alcohol, and an amino acid, wherein the potency of thebotulinum toxin is at least about 30% of the theoretical maximum potencyof the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, apolyethylene glycol, and a compound selected from the group of compoundsconsisting of a monosaccharide, a disaccharide, a trisaccharide, ametal, an alcohol, and an amino acid, wherein the potency of thebotulinum toxin is at least about 40% of the theoretical maximum potencyof the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, apolyethylene glycol, and a compound selected from the group of compoundsconsisting of a monosaccharide, a disaccharide, a trisaccharide, ametal, an alcohol, and an amino acid, wherein the potency of thebotulinum toxin is at least about 50% of the theoretical maximum potencyof the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, apolyethylene glycol, and a compound selected from the group of compoundsconsisting of a monosaccharide, a disaccharide, a trisaccharide, ametal, an alcohol, and an amino acid, wherein the potency of thebotulinum toxin is at least about 60% of the theoretical maximum potencyof the botulinum toxin.

My invention also encompasses a pharmaceutical composition comprising(or consisting of or consisting essentially of) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient, apolyethylene glycol, and a compound selected from the group of compoundsconsisting of a monosaccharide, a disaccharide, a trisaccharide, ametal, an alcohol, and an amino acid, wherein the potency of thebotulinum toxin is at least about 70% of the theoretical maximum potencyof the botulinum toxin.

DESCRIPTION

The present invention is based upon the discovery that a Clostridialtoxin pharmaceutical composition, with a stabilized Clostridial toxin,can be made using a non-protein excipient as the primary stabilizer ofthe Clostridial toxin.

I have discovered that a suitable replacement for a protein excipient,such as albumin or gelatin in a Clostridial toxin pharmaceuticalcomposition can be a non-protein compound.

The non-protein excipient used in the present invention can impartstability to a neurotoxin active ingredient, such as a botulinum toxin,present in the pharmaceutical composition by: (1) reducing adhesion(commonly referred to as “stickiness”) of the botulinum toxin tosurfaces, such as the surfaces of laboratory glassware, vessels, thevial in which the pharmaceutical composition is reconstituted and theinside surface of the syringe used to inject the pharmaceuticalcomposition. Adhesion of the botulinum toxin to surfaces can lead toloss of botulinum toxin and to denaturation of retained botulinum toxin,both of which reduce the toxicity of the botulinum toxin present in thepharmaceutical composition. (2) reducing the denaturation of thebotulinum toxin and/or dissociation of the botulinum toxin from othernon-toxin proteins present in the botulinum toxin complex, whichdenaturation and/or dissociation activities can occur because of the lowdilution of the botulinum toxin present in the pharmaceuticalcomposition (i.e. prior to lyophilization or vacuum drying) and in thereconstituted pharmaceutical composition. (3) reducing loss of botulinumtoxin (i.e. due to denaturation or dissociation from non-toxin proteinsin the complex) during the considerable pH and concentration changeswhich take place during preparation, processing and reconstitution ofthe pharmaceutical composition.

The three types of botulinum toxin stabilizations provided by thenon-protein stabilizers disclosed herein conserve and preserve thebotulinum toxin with it native toxicity prior to injection of thepharmaceutical composition.

In certain embodiments of the invention, the pharmaceutical compositionsof the invention may comprise a plurality of botulinum toxin serotypes.In other words, the composition may include two or more differentbotulinum toxin serotypes. For example, a composition may includebotulinum toxin serotypes A and B. In another embodiment, a compositionmay include botulinum toxin serotypes A and E. Using a combination ofbotulinum toxin serotypes will permit caregivers to customize thecomposition to achieve a desired effect based on the condition beingtreated. In an additional embodiment of the invention, the compositionmay comprise a modified botulinum toxin. The modified botulinum toxinwill preferably inhibit the release of neurotransmitter from a neuron,but may have a greater or lower potency than the native botulinum toxin,or may have a greater or lower biological effect than the nativebotulinum toxin. Because the compositions of the invention may be usedfor relatively long-term treatment of animals, the compositions may beprovided in a relatively pure form. In one embodiment, the compositionsare of a pharmaceutical grade. In certain embodiments, the clostridialneurotoxin has a greater than 95% purity. In additional embodiments, theclostridial neurotoxin has a purity greater than 99%.

My invention also encompasses addition of a preservative, either in thediluent or formulation itself, to allow extended storage. A preferredpreservative is preserved saline containing benzyl alcohol.

The pharmaceutical compositions of the invention can be administeredusing conventional modes of administration. In preferred embodiments ofthe invention, the compositions are administered intramuscularly orsubcutaneously to the subject. In other embodiments, the compositions ofthe invention may be administered intrathecally. In addition, thecompositions of the invention may be administered with one or moreanalgesic or anesthetic agents.

The most effective mode of administration and dosage regimen for thecompositions of this invention depends upon the type, severity, andcourse of the condition being treated, the animal's health and responseto treatment, and the judgment of the treating doctor. Accordingly, themethods and dosages of the compositions should be tailored to theindividual subject.

By way of example, and not by way of limitation, it may be preferred toadminister the composition of the invention intramuscularly to reduce amuscle spasm.

My invention also encompasses a pharmaceutical composition comprising abotulinum toxin and a collagen for use to treat a variety of conditionswherein the botulinum toxin acts to paralyze a muscle and the collagenacts to provide a dermal filler.

Compositions containing other serotypes of botulinum toxin may containdifferent dosages of the botulinum toxin. For example, botulinum toxintype B may be provided in a composition at a greater dose than acomposition containing botulinum toxin type A. In one embodiment of theinvention, botulinum toxin type B may be administered in an amountbetween about 1 U/kg and 150 U/kg. Botulinum toxin type B may also beadministered in amounts of up to 20,000 U (mouse units, as describedabove). In another embodiment of the invention, botulinum toxin types Eor F may be administered at concentrations between about 0.1 U/kg and150 U/kg. In addition, in compositions containing more than one type ofbotulinum toxin, each type of botulinum toxin can be provided in arelatively smaller dose than the dose typically used for a singlebotulinum toxin serotype. The combination of botulinum toxin serotypesmay then provide a suitable degree and duration of paralysis without anincrease in diffusion of the neurotoxins (e.g. see U.S. Pat. No.6,087,327).

EXAMPLES

The following examples set forth specific embodiments of the presentinvention and are not intended to limit the scope of the invention.

In the Examples below the well known mouse lethal dose₅₀ assay (the“MLD50 assay”) was used to determine botulinum toxin potency. Dependingon the circumstances, “potency” can mean the recovered potency of thebotulinum toxin or the potency of the botulinum toxin prior tolyophilization. Recovered potency is synonymous with reconstitutionpotency, recovery potency and with potency upon reconstitution. TheMLD50 assay provides a determination of the potency of a botulinum toxinin terms of its mouse 50% lethal dose or “LD50”. Thus, one unit (U) of abotulinum toxin is defined as the amount of botulinum toxin which uponintraperitoneal injection kills 50% (i.e. a LD₅₀) of a group of femaleSwiss Weber mice weighing 17-22 grams each at the start of the assay.The MLD50 assay is a validated method for measuring the potency of areconstituted botulinum toxin or of a reconstituted botulinum toxinformulation. Each mouse is held in a supine position with its headtilted is down and is injected intraperitoneally into the lower rightabdomen at an angle of about 30 degrees using a 25 to 27 gauge ⅜″ to ⅝″needle with one of several serial dilutions of the botulinum toxin innormal saline. The death rates over the ensuing 72 hours for eachdilution are recorded. A minimum of six dilutions at 1.33 dose intervalsare prepared and typically ten animals are used in each dosage group (60mice employed therefore). Two reference standard assays are carried outconcurrently (additional 60 mice employed). The dilutions are preparedso that the most concentrated dilution produces a death rate of at least80% of the mice injected, and the least concentration dilution producesa death rate no greater than 20% of the mice injected. There must be aminimum of four dilutions that fall within the monotone decreasing rangeof the death rates. The monotone decreasing range commences with a deathrate of no less than 80%. Within the four or more monotone decreasingrates, the two largest and the two smallest rates must be decreasing(i.e. not equivalent). The dilution at which 50% of the mice die withinthe three day post injection observation period is defined as a dilutionwhich comprises one unit (1 U) of the botulinum toxin. A refined MLD50assay has been developed which uses fewer (five instead of six)dilutions at 1.15 dose intervals and fewer mice (six instead of ten) perdilution tested.

Example 1 Botulinum Toxin Pharmaceutical Composition Containing HumanSerum Albumin (Prior Art)

A botulinum toxin type A complex was obtained from a culture of the Hallstrain of Clostridium botulinum grown in a medium containing N-Z amineand yeast extract. The botulinum toxin type A complex was purified fromthe culture solution by a series of acid precipitations to a crystallinecomplex consisting of the active high molecular weight toxin protein andan associated hemagglutinin protein. The crystalline complex was thenre-dissolved in a solution containing saline and albumin and sterilefiltered (0.2 microns) prior to vacuum-drying. The vacuum driedcomposition was reconstituted with sterile, non-preserved saline priorto injection. Each vial of vacuum dried composition contains about 100units (U) of Clostridium botulinum toxin type A complex, 0.5 milligramsof human serum albumin and 0.9 milligrams of sodium chloride in asterile, vacuum-dried form without a preservative. This pharmaceuticalcomposition is sold commercially under the trade name BOTOX® in 100 unitvials for reconstitution with saline prior to injection.

Example 2 Non-Protein Stabilized Botulinum Toxin Formulations

Experiments were carried out to prepare multiple botulinum toxinformulations with one or more different non-protein stabilizingexcipients. All of the formulations were compounded, lyophilized,reconstituted and potency assessed in the same manner, and with the sametype of botulinum toxin used in each formulation, expect that eachformulation was prepared with a different non-protein excipient orexcipients or with a different amount of the non-protein excipient or ofthe non-protein excipients present in the botulinum toxin formulation.

The non-protein excipients used (separately or in combination) in theformulations made in these experiments included: a polyvinylpyrrolidone(also called povidone, such as Kollidon 17); various disaccharides (suchas lactose and trehalose); a trisaccharide (such as raffinose); apolysaccharide (such as inulin); an alcohol (such as an alcohol made byreducing a monosaccharide [such as fructose] or such as mannitol); ametal (such as zinc); an amino acid (such as glycine), and; apolyethylene glycol (such as poloxamer 188 and/or PEG 3350). Since aprotein is a polyamino acid, use of one or more single amino acids inthe formulations set forth herein did not provide a protein excipient inthese formulations.

The formulations disclosed in this Example were made by first adding theindicated amount of the non-protein excipient(s) to sterile water forinjection to form a solution. Next between 100 to 200 units of abotulinum toxin type A complex (obtained by anaerobic fermentation ofthe Hall stain of Clostridium botulinum followed by purification of thebotulinum toxin released into and removed from the fermentation medium.See e.g. Example 1 above and Schantz E. J. et al., Properties and use ofbotulinum toxin and other microbial neurotoxins in medicine, MicrobiolRev 1992 March; 56(1):80-99) was added to the solution, to thereby forma botulinum toxin formulation, which can be synonymously be referred toas a botulinum toxin pharmaceutical composition, or simply as aformulation. The potency prior to lyophilization of the botulinum toxinused was determined by the mouse LD50 assay prior to the addition of thebotulinum toxin to the solution, and was between about 100 units andabout 200 units.

The formulations were then lyophilized (or freeze dried or vacuum dried)followed by reconstitution with normal saline. Recovered potency of thebotulinum toxin present in the reconstituted formulation was determinedby application of the same mouse LD50 assay.

“% Recovery” in Tables 1 to 5 is the potency of the botulinum toxinafter reconstitution (therefore “recovered potency”) expressed as apercent of the potency of the botulinum toxin before lyophilization ofthe formulation. Thus, for example, a % Recovery of 60% means that thepotency of the botulinum toxin after reconstitution was 60% of thepotency of the botulinum toxin prior to lyophilization. The maximumtheoretical recovered potency is 100%. The % Recovery values wereobtained by reconstitution right after the formulation was lyophilized.

Tables 1 to 6 present the results of the experiments carried out in thisExample where the formulations were made as set forth above.

TABLE 1 Botulinum Toxin Formulations with a Single Non-Protein Excipientand No Recovered Potency Excipient Amount (mg) % Recovery  1. Kollidon17 0.5 0  2. Kollidon 17 50 0  3. Kollidon 17 100 0  4. Kollidon 17 2500  5. Lactose 5 0  6. Sucrose 5 0  7. Sucrose 10 0  8. Sucrose 50 0  9.Sucrose 100 0 10. Sucrose 250 0 11. Glycine 5 0 12. Glycine 10 0 13.Glycine 50 0 14. ZnCl 0.1 0 15. ZnCl 0.01 0 16. ZnCl 0.001 0 17.Mannitol 5 0 18. Mannitol 10 0 19. Mannitol 50 0 20. Inulin 5 0 21.Inulin 10 0 22. Inulin 50 0 23. Trehalose 5 0 24. Trehalose 10 0 25.Trehalose 50 0 26. Raffinose 5 0 27. Raffinose 10 0 28. Raffinose 50 029. PEG 3350 50 0 30. Poloxamer 188 50 0

TABLE 2 Botulinum Toxin Formulations with a Single Non-Protein Excipientand a Recovered Potency Excipient Amount (mg) % Recovery 1. Kollidon 175 48 2. Kollidon 17 10 52 3. Kollidon 17 20 39 4. Lactose 10 15 5.Lactose 50 35

TABLE 3 Botulinum Toxin Formulations with a Two Non-Protein Excipientsand No Recovered Potency Amount 1 Amount 2 Excipient 1 Excipient 2 (mg)(mg) % Recovery  1. Kollidon 17 Lactose 0.5 0.5 0  2. Kollidon 17Lactose 50 0.5 0  3. Kollidon 17 Lactose 100 5 0  4. Kollidon 17 Sucrose0.5 0.5 0  5. Kollidon 17 Sucrose 50 0.5 0  6. Kollidon 17 Sucrose 0.5 50  7. Kollidon 17 Sucrose 100 5 0  8. Sucrose ZnCl 50 0.000005 0  9.Mannitol ZnCl 50 0.000005 0 10. Mannitol PEG 3350 5 50 0 11. MannitolSucrose 50 5 0 12. Mannitol Sucrose 5 50 0 13. Mannitol ZnCl 50 1 0 14.Mannitol ZnCl 50 0.1 0 15. Mannitol ZnCl 5 1 0 16. Mannitol Trehalose 5050 0 17. Mannitol Trehalose 50 5 0 18. Mannitol Trehalose 5 50 0 19.Sucrose Glycine 50 50 0 20. Sucrose Glycine 50 5 0 21. Sucrose Glycine 550 0 22. Sucrose ZnCl 50 1 0 23. Sucrose ZnCl 50 0.1 0 24. Sucrose ZnCl5 1 0 25. Sucrose Trehalose 50 50 0 26. Sucrose Trehalose 50 5 0 27.Sucrose Trehalose 5 50 0 28. ZnCl Glycine 1 50 0 29. ZnCl Glycine 1 5 030. ZnCl Glycine 0.1 50 0 31. Poloxamer 188 ZnCl 50 1 0 32. Poloxamer188 ZnCl 5 1 0 33. Trehalose ZnCl 50 1 0 34. Trehalose ZnCl 5 1 0 35.Trehalose ZnCl 50 0.1 0 36. PEG 3350 ZnCl 50 1 0 37. PEG 3350 ZnCl 500.1 0 38. Poloxamer 188 Glycine 5 50 0 39. Poloxamer 188 PEG 3350 50 500 40. Poloxamer 188 PEG 3350 50 5 0 41. Poloxamer 188 PEG 3350 5 50 042. Trehalose Glycine 50 50 0 43. Trehalose Glycine 50 5 0 44. TrehaloseGlycine 5 50 0 45. Trehalose PEG 3350 50 50 0 46. PEG 3350 Glycine 50 500 47. PEG 3350 Glycine 50 5 0 48. PEG 3350 Glycine 5 50 0

TABLE 4 Botulinum Toxin Formulations with a Two Non-Protein Excipientsand a Recovered Potency Amount 1 Amount 2 % Excipient 1 Excipient 2 (mg)(mg) Recovery  1. Kollidon 17 Lactose 5 0.5 65  2. Kollidon 17 Lactose10 0.5 47  3. Kollidon 17 Lactose 20 0.5 65  4. Kollidon 17 Lactose 0.55 52  5. Kollidon 17 Lactose 5 5 57  6. Kollidon 17 Lactose 10 5 65  7.Kollidon 17 Lactose 20 5 49  8. Kollidon 17 Lactose 50 5 52  9. Kollidon17 Sucrose 5 0.5 58 10. Kollidon 17 Sucrose 10 0.5 46 11. Kollidon 17Sucrose 20 0.5 49 12. Kollidon 17 Sucrose 5 5 49 13. Kollidon 17 Sucrose10 5 58 14. Kollidon 17 Sucrose 20 5 47 15. Kollidon 17 Sucrose 50 5 3916. Kollidon 17 Sucrose 250 250 39 17. Kollidon 17 Sucrose 10 250 58 18.Kollidon 17 PEG 3350 50 50 35 19. Lactose PEG 3350 50 50 53 20. LactoseSucrose 50 50 27 21. Lactose ZnCl 50 0.000005 19 22. Lactose Mannitol 5050 23 23. Sucrose Mannitol 50 50 24 24. Mannitol PEG 3350 50 50 26 25.Mannitol PEG 3350 50 5 30 26. Mannitol Sucrose 50 50 29 27. MannitolPoloxamer 50 50 33 188 28. Mannitol Poloxamer 50 5 35 188 29. MannitolPoloxamer 5 50 30 188 30. Sucrose Poloxamer 50 50 59 188 31. SucrosePoloxamer 50 5 43 188 32. Sucrose Poloxamer 5 50 55 188 33. Sucrose PEG3350 50 50 44 34. Sucrose PEG 3350 50 5 41 35. Sucrose PEG 3350 5 50 3536. Poloxamer 188 ZnCl 50 0.1 38 37. PEG 3350 ZnCl 5 1 23 38. Poloxamer188 Glycine 50 50 26 39. Poloxamer 188 Glycine 50 5 33 40. Poloxamer 188Trehalose 50 50 53 41. Poloxamer 188 Trehalose 50 5 75 42. Poloxamer 188Trehalose 5 50 50 43. Trehalose PEG 3350 50 5 41 44. Trehalose PEG 33505 50 36

Table 5 shows the results of experiments with botulinum toxinformulations containing two non-protein excipients and with or withoutthe specified buffer. The potency of the botulinum toxin was measured:(1) after lyophilization and immediate reconstitution (“InitialPotency”), and; (2) after lyophilization, storage for three months underone of two different storage conditions (at −40 degrees C. or at 20degrees C.) and reconstitution.

The Table 5 results show that a botulinum toxin pharmaceuticalcomposition containing a PVP non-protein excipient does not havesignificant room temperature stability unless formulated with a citratebuffer. I found that even at the same pH a phosphate buffer would notprovide the desired room temperature stability for such a formulation.

TABLE 5 Botulinum Toxin Formulations with Two Non-Protein ExcipientsPotency Potency Potency (3 Month (3 Month Room Non-Protein Excipients(Initial) Freezer) Temperature) 20 mg sucrose 101% 117% 87% 10 mgpoloxamer 20 mg sucrose 77% 81% 81% 10 mg poloxamer 10 mM citrate (pH5.5) 20 mg sucrose 112% 113% 113% 10 mg poloxamer 10 mM phosphate (pH5.5) 20 mg sucrose 90% Not obtained 91% 10 mg poloxamer 10 mM citrate(pH 6.5) 20 mg sucrose 95% 119% 88% 10 mg poloxamer 10 mM phosphate (pH6.5) 20 mg PVP 71% 101% <39% 10 mg poloxamer 20 mg PVP 65% 101% 65% 10mg poloxamer 10 mM citrate (pH 5.5) 20 mg PVP 71% 79% <39% 10 mgpoloxamer 10 mM phosphate (pH 5.5) 20 mg PVP 87% 97% <39% 10 mgpoloxamer 10 mM citrate (pH 6.5) 20 mg PVP 65% 63% <39% 10 mg poloxamer10 mM phosphate (pH 6.5)

The Table 6 shows the results of experiments with botulinum toxinformulations containing three non-protein excipients and with or withoutthe specified buffer. The potency of the botulinum toxin was measuredafter lyophilization followed by immediate reconstitution (“InitialPotency”).

The Table 6 results show that a botulinum toxin pharmaceuticalcomposition can be stabilized by use of three non-protein excipientpresent in the same formulation, and that the use of a citrate buffer inthe formulation in the formulation improves initial potency. Botulinumtoxin pharmaceutical compositions stabilized with other three differentnon-protein excipients, and with significant recovered potency, werealso made.

TABLE 6 Botulinum Toxin Formulations with Three Non-Protein ExcipientsNon-Protein Excipients Potency (Initial) 20 mg sucrose 67% 20 PVP 20 mgpoloxamer 20 mg sucrose 98% 20 mg PVP 20 mg poloxamer 10 mM citrate (pH5.5)

Among the discoveries made from these experiments were the following:

1. a botulinum toxin formulation prepared with particular concentrationsof a single non-protein excipient which is a polyvinylpyrrolidone (suchas Kollidon 17) can show no recovered potency (see lines 1-4 of Table1).

2. a botulinum toxin formulation prepared with different particularconcentrations of a single non-protein excipient which is apolyvinylpyrrolidone (such as Kollidon 17) can show a recovered potencybetween 39% and 52% (see lines 1-3 of Table 2). In light of item 1.above this is a surprising and unexpected discovery.

3. a botulinum toxin formulation prepared with a two differentnon-protein excipients, wherein one of the non-protein excipients is apolyvinylpyrrolidone (such as Kollidon 17) can show no recovered potency(see lines 1-7 of Table 3).

4. a botulinum toxin formulation prepared with a two differentnon-protein excipients, wherein one of the non-protein excipients is apolyvinylpyrrolidone (such as Kollidon 17) can show a recovered potencyas high as 65% (see lines 1-18 of Table 4). In light of items 1 and 3.above this is a surprising and unexpected discovery.

5. a botulinum toxin formulation prepared with a particularconcentration of a single non-protein excipient which is a disaccharide(such as lactose) can show no recovered potency (see line 5 of Table 1).

6. a botulinum toxin formulation prepared with different particularconcentrations of a single non-protein excipient which is a disaccharide(such as lactose) can show a recovered potency between 15% and 35% (seelines 4-5 of Table 2). In light of item 5. above this is a surprisingand unexpected discovery.

7. a botulinum toxin formulation prepared with a two differentnon-protein excipients, wherein one of the non-protein excipients is adisaccharide (such as lactose) can show no recovered potency (see lines1-3 of Table 3).

8. a botulinum toxin formulation prepared with a two differentnon-protein excipients, wherein one of the non-protein excipients is ais a disaccharide (such as lactose) can show a recovered potency as highas 65% (see lines 1-8 and 19-22 of Table 4). In light of items 5 and 7.above this is a surprising and unexpected discovery

9. a botulinum toxin formulation prepared with a single non-proteinexcipient which is a disaccharide (such as sucrose) can show norecovered potency (see lines 6-10 of Table 1).

10. a botulinum toxin formulation prepared with a two differentnon-protein excipients, wherein one of the non-protein excipients is adisaccharide (such as sucrose) can show no recovered potency (see lines4-8, 11-12, and 19-27 of Table 3).

11. a botulinum toxin formulation prepared with two differentnon-protein excipients, wherein one of the non-protein excipients is adisaccharide (such as sucrose) can show a recovered potency as high as59% (see lines 9-17, 20, 23, 26 and 30-35 of Table 4). In light of items9. and 10 above this is a surprising and unexpected discovery.

12. a botulinum toxin formulation prepared with a single non-proteinexcipient which is an amino acid (such as glycine) can show no recoveredpotency (see lines 11-13 of Table 1).

13. a botulinum toxin formulation prepared with a two differentnon-protein excipients, wherein one of the non-protein excipients is aamino acid (such as glycine) can show no recovered potency (see lines19-21, 28-30, 38, 42-44 and 46-48 of Table 3).

14. a botulinum toxin formulation prepared with different particularconcentrations of two different non-protein excipients, wherein one ofthe non-protein excipients is an amino acid (such as glycine) can show arecovered potency as high as 33% (see lines 38-39 of Table 4). In lightof items 12-13 above this is a surprising and unexpected discovery.

15. a botulinum toxin formulation prepared with a single non-proteinexcipient which is a metal (such as zinc) can show no recovered potency(see lines 14-16 of Table 1).

16. a botulinum toxin formulation prepared with a two differentnon-protein excipients, wherein one of the non-protein excipients is ametal (such as zinc) can show no recovered potency (see lines 8-9,13-15, 22-24, and 28-37 of Table 3).

17. a botulinum toxin formulation prepared with different particularconcentrations of two different non-protein excipients, wherein one ofthe non-protein excipients is a metal (such as zinc) can show arecovered potency as high as 38% (see lines 21 and 36-37 of Table 4). Inlight of items 15-16 above this is a surprising and unexpecteddiscovery.

18. a botulinum toxin formulation prepared with a single non-proteinexcipient which is an alcohol (such as mannitol) can show no recoveredpotency (see lines 17-19 of Table 1).

19. a botulinum toxin formulation prepared with a two differentnon-protein excipients, wherein one of the non-protein excipients is analcohol (such as mannitol) can show no recovered potency (see lines 9-18of Table 3).

20. a botulinum toxin formulation prepared with different particularconcentrations of two different non-protein excipients, wherein one ofthe non-protein excipients is an alcohol (such mannitol) can show arecovered potency as high as 35% (see lines 22-29 of Table 4). In lightof items 17-18 above this is a surprising and unexpected discovery.

21. a botulinum toxin formulation prepared with a single non-proteinexcipient which is a disaccharide (such as trehalose) can show norecovered potency (see lines 23-25 of Table 1).

22. a botulinum toxin formulation prepared with a two differentnon-protein excipients, wherein one of the non-protein excipients is adisaccharide (such as trehalose) can show no recovered potency (seelines 16-18, 25-27, 33-35 and 42-45 of Table 3).

23. a botulinum toxin formulation prepared with different particularconcentrations of two different non-protein excipients, wherein one ofthe non-protein excipients is a disaccharide (such as trehalose) canshow a recovered potency as high as 75% (see lines 40-44 of Table 4). Inlight of items 21-22 above this is a surprising and unexpecteddiscovery.

24. a botulinum toxin formulation prepared with a single non-proteinexcipient which is a polyethylene glycol (such as PEG 3350 or poloxamer188) can show no recovered potency (see lines 29-30 of Table 1).

25. a botulinum toxin formulation prepared with a two differentnon-protein excipients, wherein one of the non-protein excipients is apolyethylene glycol (such as PEG 3350 or poloxamer 188) can show norecovered potency (see lines 10, 31-32, 36-41 and 45-48 of Table 3).

26. a botulinum toxin formulation prepared with different particularconcentrations of two different non-protein excipients, wherein one ofthe non-protein excipients is a polyethylene glycol (such as PEG 3350 orpoloxamer 188) can show a recovered potency as high as 75% (see lines18-19, 24-25, 27-44 of Table 4). In light of items 24-25 above this is asurprising and unexpected discovery.

27. The non-protein stabilizers lactose and polyvinylpyrrolidone (“PVP”)(i.e. Kollidon 17) each provided significant recovery potency when usedas a non-protein stabilizer of the botulinum toxin present in abotulinum toxin pharmaceutical composition (see Table 2).

28. When lactose and PVP used were both used as non-protein stabilizersof the same botulinum toxin pharmaceutical composition the recoverypotency improved, as compared to the recovery potency observed when thelactose and PVP where used separately as a non-protein stabilizer (seeeg Table 4, lines 1-8).

29. Recovery potency (of the botulinum toxin present in a reconstitutedbotulinum toxin pharmaceutical composition) improved when lactose and/orPVP were used with one or more of the other non-protein excipients setforth above (see, respectively, Table 4, lines 19-22, and Table 4, lines9-18).

30. Use of certain combinations of excipients (as non-proteinstabilizers of the botulinum toxin present in a reconstituted botulinumtoxin pharmaceutical composition) provided a significant recoverypotency even where no recovery potency was obtained when either suchnonprotein excipient was used by itself as a non-protein stabilizer ofthe botulinum toxin present in a botulinum toxin pharmaceuticalcomposition. For example, compare: (1) Table 1, lines 1-4 and Table 1,lines 6-10, with Table 4, lines 9-17, and; (2) Table 1, line 23-25 andTable 1, line 29, with Table 4, 43-44.

31. Recovery potency was sometimes dependent upon the concentration ofthe non-protein stabilizer present in the botulinum toxin pharmaceuticalcomposition

32. Addition of a buffer could improve recovery potency and storagestability. Individual buffers differed in their capacity to exert thiseffect. The buffers act to obtain optimal pH, maintain optimal pH, andin some instances (e.g., citrate) to protect against oxidation.

General conclusions from these experiments include the observationsthat:

(a) The botulinum toxin present in a botulinum toxin pharmaceuticalcomposition can be stabilized (and shown by a good recovery potency) byformulating the composition with two or more common non-proteinexcipients.

(b) a polyvinylpyrrolidone (such as Kollidon 17) and a disaccharide(such as lactose) can function as a non-protein stabilizer (excipient)in a botulinum toxin formulation without the presence of any othernon-protein stabilizer.

(c) with the same non-protein stabilizer or stabilizers, recoveredpotency can be dependent upon the ratio of and/or the concentration ofthe non-protein stabilizer or stabilizers used in the botulinum toxinformulation.

(d) certain non-protein stabilizers (such as a polyvinylpyrrolidone[such as Kollidon 17] and a disaccharide [such as lactose]) not only canact to stabilize the botulinum toxin in a botulinum toxin formulationwhen used together, but can provide an enhanced stabilization when usedtogether, as determined by a higher recovered potency of thereconstituted formulation.

(e) commonly used pharmaceutical excipients (such aspolyvinylpyrrolidone, lactose, sucrose etc) did not function when usedas stabilizers, or only function as a stabilizer of a botulinum toxin ina non-protein botulinum toxin formulation when used at particularconcentrations. a polyvinylpyrrolidone (such as Kollidon 17) and adisaccharide (such as lactose)

(f) Many excipients functioned or functioned as better stabilizers of abotulinum toxin in a non-protein botulinum toxin formulation whencombined with a polyvinylpyrrolidone (such as Kollidon 17) or with adisaccharide (such as lactose).

(g) Although the specific PVP “Kollidon 17” was used in a number of thebotulinum toxin formulations made other PVPs are within the scope of thepresent invention.

(h) Although the specific poloxamer “polyoxamer 188” was used in anumber of the botulinum toxin formulations made other poloxamers arewithin the scope of the present invention.

(i) It was found that the surfactant polysorbate (Tween) can be usedinstead of poloxamer 188 with similar results.

Other non-protein excipients which can be used in a botulinum toxinpharmaceutical composition within the scope of the present inventioninclude antioxidants such as butylated Hydroxytoluene (BHT) andbutylated Hydroxyanisole (BHA) and amino acids such as cysteine andmethionine. The lyophilized botulinum toxin formulation can bereconstituted with saline, water or with a custom diluent to affect theperformance after reconstitution or injection.

Example 3 Use of a Botulinum Toxin Pharmaceutical Composition

A 48 year old male is diagnosed with a spastic muscle condition, such ascervical dystonia. Between about 10⁻³ U/kg and about 35 U/kg of abotulinum toxin type A pharmaceutical composition of formulationcontaining lactose and PVP is injected intramuscularly into the patient.Within 1-7 days the symptoms of the spastic muscle condition arealleviated and alleviation of the symptoms persists for at least fromabout 2 months to about 6 months.

A pharmaceutical composition according to the invention disclosed hereinhas many advantages, including the following:

1. the pharmaceutical composition can be prepared free of any bloodproduct, such as albumin and therefore free of any blood productinfectious element such as a prion.

2. the pharmaceutical composition has stability and high % recovery oftoxin potency comparable to or superior to that achieved with currentlyavailable pharmaceutical compositions.

3. reduced toxicity, as assessed by either intramuscular or intravenousadministration.

4. reduced antigenicity.

Various publications, patents and/or references have been cited herein,the contents of which, in their entireties, are incorporated herein byreference.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of stabilizing polysaccharides and aminoacids are within the scope of the present invention.

Accordingly, the spirit and scope of the following claims should not belimited to the descriptions of the preferred embodiments set forthabove.

1. A pharmaceutical composition comprising: (a) a botulinum toxin,wherein the botulinum toxin is not stabilized by a protein excipient,(b) a polyvinylpyrrolidone, and; (c) a disaccharide, wherein the potencyof the botulinum toxin is at least about 40% of the theoretical maximumpotency of the botulinum toxin.
 2. The pharmaceutical composition ofclaim 1, wherein the botulinum toxin is selected from the groupconsisting of the botulinum toxins types A, B, C, D, E, F and G.
 3. Thepharmaceutical composition of claim 1, wherein the botulinum toxin is abotulinum toxin type A.
 4. A pharmaceutical composition comprising: (a)a botulinum toxin, wherein the botulinum toxin is not stabilized by aprotein excipient, (b) a polyvinylpyrrolidone, and; (c) a disaccharide,wherein the potency of the botulinum toxin is at least about 50% of thetheoretical maximum potency of the botulinum toxin.
 5. Thepharmaceutical composition of claim 4, wherein the botulinum toxin isselected from the group consisting of the botulinum toxins types A, B,C, D, E, F and G.
 6. The pharmaceutical composition of claim 4, whereinthe botulinum toxin is a botulinum toxin type A.
 7. A pharmaceuticalcomposition comprising: (a) a botulinum toxin, wherein the botulinumtoxin is not stabilized by a protein excipient, (b) apolyvinylpyrrolidone, and; (c) a disaccharide, wherein the potency ofthe botulinum toxin is at least about 60% of the theoretical maximumpotency of the botulinum toxin.
 8. The pharmaceutical composition ofclaim 7, wherein the botulinum toxin is selected from the groupconsisting of the botulinum toxins types A, B, C, D, E, F and G.
 9. Thepharmaceutical composition of claim 7, wherein the botulinum toxin is abotulinum toxin type A.
 10. A pharmaceutical composition comprising: (a)a botulinum toxin, wherein the botulinum toxin is not stabilized by aprotein excipient, (b) a polyvinylpyrrolidone, and; (c) a disaccharide,wherein the potency of the botulinum toxin is at least about 70% of thetheoretical maximum potency of the botulinum toxin.
 11. Thepharmaceutical composition of claim 10, wherein the botulinum toxin isselected from the group consisting of the botulinum toxins types A, B,C, D, E, F and G.
 12. The pharmaceutical composition of claim 10,wherein the botulinum toxin is a botulinum toxin type A.